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Hemostasis
Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura
Ferrata Storti Foundation
Haematologica 2020 Volume 105(11):2619-2630
Nuno A. G. Graça,1,2 Bogac Ercig,2,3,4 Leydi Carolina Velásquez Pereira,5 Kadri Kangro,5 Paul Kaijen,2 Gerry A. F. Nicolaes,3,4 Agnès Veyradier,6,7 Paul Coppo,7-9 Karen Vanhoorelbeke,5 Andres Männik1 and Jan Voorberg2
1Icosagen Cell Factory OÜ, Õssu, Kambja, Tartumaa, Estonia; 2Department of Molecular and Cellular Hemostasis, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, the Netherlands; 3Pharmatarget, Maastricht, the Netherlands; 4Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands; 5Laboratory for Thrombosis Research, IRF Life Sciences, KU, Leuven Campus Kulak Kortrijk, Kortrijk, Belgium; 6Service d’Hématologie Biologique and EA3518‐Institut Universitaire d’Hématologie, Groupe Hospitalier Saint Louis-Lariboisière, AP-HP, Université Paris Diderot, Paris, France; 7Centre de Référence des Microangiopathies Thrombotiques, Hôpital Saint-Antoine, AP-HP, Paris, France; 8Service d’Hématologie, Hôpital Saint-Antoine, AP-HP, Paris, France and 9Sorbonne Université, UPMC Université Paris, Paris, France
ABSTRACT
Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY) of ADAMTS13 (a disinte- grin and metalloproteinase with a thrombospondin type 1 motif, mem- ber 13). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced to create a large collection of full-length ADAMTS13 variants comprising conservative (Y←→F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported gain-of-function (KYKFF) and truncated ‘MDTCS’ variants were also included. Sera from 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues showed a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine muta- tions of aromatic residues were the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoanti- bodies targeting the spacer RFRYY epitope were preponderant compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest reductions in activity were observed in the most autoantibody- resistant variants (15-35% residual activity in a FRETS-VWF73 assay). Among these, a triple-alanine mutant – RARAA – showed activity in a von Willebrand factor multimer assay. This study shows that non-con- servative and alanine modifications of residues within the exosite-3 spac- er RFRYY epitope in full-length ADAMTS13 resist the binding of autoan- tibodies from patients with immune thrombotic thrombocytopenic pur- pura, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.
Partially presented as an oral communication at the 9th Bari International Conference, Rome, Italy, September 17, 2017, and presented at the 10th Bari International Conference, Genoa, Italy, September 8, 2019.
Correspondence:
JAN VOORBERG
j.voorberg@sanquin.nl
Received: May 8, 2019.
Accepted: November 21, 2019. Pre-published: November 21, 2019.
doi:10.3324/haematol.2019.226068 ©2020 Ferrata Storti Foundation
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